Molecular evolution of miR159 family and their expression profiling during dormancy of Pyrus pyrifolia in the winter

Abstract:【Objective】Pear and other deciduous fruit trees have low temperature dormancy in winter. The study and elucidation of the mechanism of winter dormancy and release of flower buds of southern  early-ripening pears can provide reference for pear production in southern China. GA is an important hormone for plant growth and development and plays an important role in regulating dormancy. MiR-NAs have important roles in plant growth and development. MiR159 is a widely present and conserved miRNA that targets and regulates multiple MYB genes. MYB genes are induced by gibberellin (GA) and involved in the GA signaling pathway. This study explored the evolutionary characteristics of miR159 gene family and its expression pattern during the dormancy process of pear.【Methods】In this paper,two mature and two precursor sequences of miR159 family were obtained from miRNA library of Huan-hua pear. They were ppy-miR159a, ppy-miR159b, ppy-MIR159a and ppy-MIR159b. ppy-miR159a and ppy-miR159b were cloned from the bud of Huanghua. Changes in these two pear miR159 members and their target gene pyrus_GLEAN_10035941 (GAMYB transcription factor) during the process of pear flower bud dormancy were determined by real-time PCR analysis. In the treatment group, 1% cyana-mide (HC) was used to treat the pear branches during endodormancy period, and spraying water was used for the control group. The two miR159 family members and their target gene pyrus_GLEAN_10035941(GAMYB transcription factor) were determined. expression changes. The role of miR159 family in pear flower bud dormancy process was analyzed.【Results】The results of the phylogenetic tree of the mature miR159 family in plants showed that in the large branch of the phylogenetic tree, the base se-quence of the miR159 family were highly overlapped, and most of the miR159 families had the same se-quence, suggesting that miR159 is highly conserved in the evolutionary process. Various small branches appeared in the evolutionary tree, indicating that the miR159 family also had some base changes in its mature sequence due to functional differentiation during the evolution process. The precursors of both miR159 family members can form a ring structure, and the mature bodies are both at the 3' arm. The minimum folding free energy (ΔG) of different members of the miR159 family measured by RNAfold was -88.15 kcal·mol^-1 and -84.36 kcal·mol^-1, respectively, indicating that the members of the miR159 family are structurally stable. Phylogenetic analysis found that the affinity of miR159 of pear was more closely related to apple. The mature sequence of the miR159 family member in 'Huanghua' pear is high-ly conserved, with changes only at position 1 and 20. PsRNA target prediction showed that potential tar-get genes of miR159 were MYB family genes, which are related to the regulation of GA. Other potential target genes of the two miR159s included cytochrome and protein kinase and were not exactly the same. qRT-PCR analysis showed that a dynamic change pattern of“decline-rise-decline”in miR159 expres- sion was observed during the dormancy process of pear bud. The results showed a downward trend in general, and the expression levels in S3 and S5 stages were significantly reduced. The expression trend of the target gene pyrus_GLEAN_10035941 is opposite to that of miR159, which was in a“rise-decline- rise”pattern. It is speculated that miR159 may play a role in regulating the dormancy process of pear flower buds by regulating the expression of GAMYB- related target gene pyrus_GLEAN_10035941. The budbreak rate in the group treated with HC was 93.05% , while that in the control group was 10.48% . The expression of miR159 was decreased and its target gene pyrus_GLEAN_10035941 in-creased by HC treatment.【Conclusion】miR159 contained two mature forms, i.e. ppy- miR159a/b in pear. The target genes of the ppy-miR159a/b was mainly MYB family genes. The release of winter dor- mancy in pear flower buds may be promoted with down- regulating miR159 and upregulating of GAMYB and by GA application. The possible mechanism of miR159 regulating pear flower bud dorman-cy process via regulating GA signaling pathway needs to be further studied. In addition, the expression patterns of target genes, pyrus_GLEAN_10035941 and DELLA, are similar, and whether there is a regu-latory relationship between these two genes and miR159 is worth exploring in subsequent studies.